Reply to ‘Double-outlet right ventricle is not hypoplastic left heart syndrome’

Our documentation of the presence of hypoplastic left heart syndrome (HLHS) in mutant mice was based on three levels of analyses¹. First, fetal echocardiography was performed, which yielded evidence of HLHS on the basis of the combination of 2D and color-flow imaging and hemodynamic assessments (Fig. 1a,b and Supplementary Videos 1 and 2 in ref. ¹). Second, necropsy was performed, which provided direct visualization of the left ventricle (LV) and aorta (Fig. 1c and Supplementary Video 3 in ref. ¹). Third, confocal histopathology was carried out with episcopic confocal microscopy for 3D reconstructions to assess intracardiac anatomy (valves and inflow/outflow connections) (Fig. 1d and Supplementary Video 4 in ref. ¹). Hence, the diagnosis of HLHS in each mutant was based on a thorough three-prong analysis: functional analysis with fetal echocardiography, and anatomical analysis with necropsy and 3D reconstructions.

We have no disagreement with the definition of HLHS described by Chaudhry et al.². Indeed, the mutants from the Ohia mouse line recovered from our screen precisely fit this classic definition of HLHS. Thus, in the data in Table 1 of ref. ¹, 23 (82%) of the 28 HLHS mutants recovered from the Ohia-mutant line had classic HLHS as defined above. We also duly noted (footnote ‘a’ in Table 1) that 5 of the 28 mutants had double-outlet right ventricle (DORV) in conjunction with hypoplastic LV. We further observed classic HLHS and also DORV with hypoplastic LV in the Sap130; Pcdha9 CRISPR-mutant mice, thus confirming that both phenotypes are heritable and are elicited by the Sap130; Pcdha9 mutations. Consequentially, these genetic findings challenge the definition of HLHS based solely on cardiac morphology.

The heterogeneity of cardiac phenotypes in genetic studies of congenital heart disease (CHD) has been challenging. We note that the phenotypic variation observed in Ohia mutants is on par with the spectrum of CHD phenotypes observed in both humans and knockout or mutant mice with CHD. Clinical studies have shown that HLHS is heritable with a variable phenotype in families. Thus, it is clinically well recognized that various forms of left-sided obstructive lesions, including HLHS, can run in families and that the phenotype can be variable even between identical twins. For example, in a study by Hinton et al.³ of families ascertained on the basis of a proband with HLHS, monozygotic twins have been observed with one twin having HLHS and the other having bicuspid aortic valve. Below, we provide a few examples in the literature reporting variability in the specific CHD phenotypes comprising persistent truncus arteriosus and DORV in mice with mutations in Pax3 (also known as Splotch), Cited2, Foxp1, and Fgf8 (refs. ^4–8).

As noted above, HLHS was shown by the images provided in Fig. 1 of ref. ¹ and was further supported by the associated supplementary videos of functional assessments by fetal echocardiography and follow-up anatomical analysis by 3D confocal histopathology. Together, these functional and anatomical assessments provide a definitive diagnosis of HLHS.

Additional documentation of the variable phenotypes is curated in the Mouse Genome Informatics database, including representative images of our mutant lines. (http://www.informatics.jax.org/allele/key/814687 for line 464; http://www.informatics.jax.org/allele/key/816745 for line 635; http://www.informatics.jax.org/allele/key/822794 for line 1430; http://www.informatics.jax.org/allele/key/822691 for line 1432; http://www.informatics.jax.org/allele/key/827867 for line 1963; http://www.informatics.jax.org/allele/key/859990 for line 3077; http://www.informatics.jax.org/allele/key/820915 for line 1709; and http://www.informatics.jax.org/allele/key/878328 for line 3183).

These combined analyses, comprising anatomical and functional assessments, showed that six of the mutant lines have a classic HLHS phenotype (lines 464, 635, 1430, 1432, 1963, and 3077), and two lines (1709 and 3183) exhibit HLHS variant phenotypes. Our CRISPR mutants can yield a classic HLHS phenotype as well as a DORV variant of HLHS, as observed in the original Ohia HLHS mutant line recovered from the screen. A CRISPR mutant with a classic HLHS phenotype is demonstrated in Supplementary Fig. 7 in ref. ¹, mutant CC-74.

Chaudhry et al. raise concerns regarding a lack of homologous protocadherin mutations between mice and humans. Given our small cohort size and that our data support multigenic inheritance, the recovery of SAP130 with a PCDHA9 mutation may be an unrealistic expectation. However, this question does highlight the difficulties in determining gene homologs across species.

In summary, our study identified genetically altered mice, the Ohia-mutant line, that have classic HLHS. Unexpectedly, in the Ohia-mutant line, we found that littermates with the same genetic alterations as mice with classic HLHS have other forms of CHD that include some features of HLHS as well as features of DORV, thus challenging the current nomenclature contained in ICD-11. Although rigid phenotype definitions may be essential to guide clinical care and surgical strategies, the findings in our study illuminate their shortcomings when trying to define the developmental and genetic origins of CHD.